Recombinant adeno-associated virus (rAAV) has several characteristics that underscore its potential as a gene therapy vector for numerous target organs and inherited diseases. rAAV vector systems potentially offer major advantages over adenovirus and retroviruses. These include the ability of rAAV to integrate into the genome of non-dividing cells, the lack of potential immune responses since all viral genes can be deleted, and the fact that rAAV can be concentrated to high titers.
Adeno-associated virus type-2 (AAV-2) has been suggested to be a very promising vector for the gene therapy of cystic fibrosis (Conrad et. al., 1996; Flotte et al., 1993; Halbert et al., 1997). In vivo administration of AAV vectors to the airway of rabbits and rhesus macaques has demonstrated long-term persistence, with viral DNA lasting up to 6 months (Conrad et al., 1996; Halbert et al., 1997). Despite the fact that this vector system has been claimed to have a very broad host tropism in a variety of human, simian, and rodent cell lines (Lebkowski et al., 1988; Muzyczka, 1992), the overall transduction efficiency in human airway epithelia seems to be quite low. In the absence of external stimuli, such as DNA damaging agents, topoisomerase inhibitor, or adenoviral early gene products (Alexander et al., 1997; Alexander et al., 1996; Ferrari et al., 1996; Fisher et al., 1996; Halbert et al., 1997; Russel et al., 1995), the in vitro transduction of primary cells and other non-dividing cells with rAAV is very inefficient. In contrast, rAAV transduction of actively dividing cells in S-phase is much more efficient (Russel et al., 1994). However, rAAV exhibits remarkable efficiency in the in situ transduction of skeletal muscle and CNS neurons, indicating that rAAV vectors can effectively transduce certain populations of non-dividing cells, and that cell-specific characteristics have profound effects on viral processing (Duan et al., 1998; Kaplitt et al., 1994; Xiao et al., 1996).
Two studies have suggested that single strand to double strand conversion of the viral genome may be the rate-limiting step for AAV-mediated gene transfer (Ferrari et al., 1996; Fisher et al., 1996). These studies demonstrated that adenovirus E4orf6 enhances the conversion of single-stranded DNA genomes to linear, double-stranded replication form dimers (Rfd) and monomers (Rfm), through a pathway characteristic of the lytic phase of rAAV replication. The structure of these replication forms consists of head-to-head and tail-to-tail orientated linear concatamers with one covalently linked end (Ferrari et al., 1996; Fisher et al., 1996). In contrast, recent studies have elucidated an alternative pathway for the conversion of rAAV genomes to double-stranded circular intermediates with head-to-tail monomer and concatamer structures (Duan et al., 1999; Duan et al., 1998; Sanlioglu et al., 1999). The distinct pathways leading to the formation of either circular AAV genomes or Rf intermediates appear to be regulated by different cellular factors. For example, adenoviral E4orf6 expression decreases circular genome formation while adenovirus E2a enhances its formation (Duan et al., 1999). Similarly, UV irradiation also enhances AAV circular intermediate formation but not Rf intermediates (Sanlioglu et al., 1999). Based on findings that circular AAV intermediates are associated with long-term episomal persistence and transgene expression in muscle (Duan et al., 1998), and UV irradiation increases both circular intermediates and the extent of integration, AAV circular intermediates may be latent phase preintegration structures.
The human airway is lined by specialized ciliated and non-ciliated epithelial cells, which not only provide protection from the external environment, but also perform functions involved in regulating the exchange of molecules between the airway lumen and underlying submucosa. These cellular functions are supported by a highly polarized organization with respect to the distribution of membrane proteins and subcellular organelles (Rodriguez-Boulan et al., 1993; Wills et al., 1996). Previous studies have suggested that this asymmetric spatial organization has significant influences on the efficiency of gene transfer. For example, the lack of integrins and adenoviral fiber receptor on the apical surface may explain the inefficient infection of differentiated, ciliated airway epithelia with this viral system (Goldman eta al., 1995; Zabner et al., 1997). Recent studies on polarized airway epithelial cells have also revealed a similar sidedness to retroviral infectivity, which may in part be explained by the partitioning of retroviral receptors to the basolateral membrane (Wang et al., 1998). In addition, similar findings of polarity in rAAV infection of polarized airway epithelia have been reported (Duan et al., 1998). These studies demonstrate a 200-fold greater infectivity of rAAV from the basolateral sides of airway epithelia.
Polarized entry into epithelial cells is also a well-known phenomenon for a variety of other viruses. For example, vaccinia virus, vesicular stomatitis virus, cytomegalovirus, canine parvovirus (CPV), and Semliki forest virus transduce polarized epithelia predominantly through basolateral membranes (Basak et al., 1989; Fuller et al., 1984; Rodriguez et al., 1991; Tugizov et al; 1996). In contrast, simian virus 40 and measles virus preferentially infect via the apical membranes (Blau et al., 1995; Clayspri et al., 1988). It is generally believed that the asymmetric distribution of cellular membrane receptors is responsible for the polarity of infection exhibited by these viruses.
However, it is also plausible that other rate limiting steps may play a role in the overall efficiency of viral transduction. These steps include virus binding, endocytosis, endosome processing, nuclear transport, uncoating, gene conversion, transcription, and translation. In this regard, previous studies in polarized MDCK cells have demonstrated a slower maturation of coated pits from the apical surface, indicating a difference in the rate of endocytosis between the apical and basolateral membranes (Naim et al., 1995).
For AAV, two approaches have been used to enhance rate-limiting steps in viral vector transduction. These include manipulation of cell surface receptors (Qing et al., 1997) and/or receptor ligands in the virus coat proteins (Wickham et al., 1996a; Wickham et al., 1996b; Bartlett et al., 1999). Alternative approaches have attempted to increase transgene expression by enhancing the molecular conversion of nonfunctional viral genomes to expressible forms in the case of rAAV (Fisher et al., 1996; Sanlioglu et al., 1999) or by increasing transcription and translation efficiencies by altering the transgene expression cassettes (Zabner et al., 1996; Xiao et al., 1998).
Cystic fibrosis is the most common inherited disease in the Caucasian population, and it is likely that gene therapy for this disorder will target the lung airway epithelium. The development of AAV as a gene therapy vehicle for treating cystic fibrosis has several unique advantages based on its viral biology. For example, wild type AAV infections are known to occur in the respiratory epithelium but have no known associated pathology. However, as described above, fully differentiated airway epithelia are extremely resistant to infection from the apical surface not only with rAAV-2 but also all other types of viral vectors currently in use, viral vectors including, adenovirus, lentivirus, retrovirus, and AAV.
Therefore, what is needed is the identification of agents which can alter, e.g., increase or enhance, rAAV transduction in vivo. What is also needed is the identification of agents that increase or enhance the expression of a transgene in rAAV in non-dividing cells such as those in the liver and the airway.